1. Field of the Invention
The present invention relates to novel methods and media for preparing bulk cultures to be used as inoculants in making fermented dairy products and further relates to novel methods for making fermented dairy products. In particular, the present invention relates to methods and media for preparing bulk cultures of proteinase negative lactic bacteria and to methods for making fermented dairy products, such as Cheddar and other cheeses, cottage cheese, cream cheese, buttermilk, and sour cream, employing such proteinase negative lactic bacteria.
2. The Prior Art
Although the present invention relates to methods for making many different kinds of fermented dairy products, the following discussion of the present invention, as well as the prior art, is generally in terms of cheesemaking. However, since the processes for making other fermented dairy products are closely akin to the processes for cheesemaking, it will be readily understood that the description of the present invention also pertains to the processes for making the other fermented dairy products, unless otherwise indicated.
Cheese is made by the controlled coagulation and syneresis of milk. Each year, the cheesemaking industry in the United States consumes literally billions of gallons of milk for the production of cheese. Coagulation and syneresis of the milk is accomplished by an extract containing an enzyme known as rennin or chymosin, which enzyme is extracted from the fourth or true stomach of a calf. (Other suitable enzyme-containing extracts obtained from bovine, swine, and fungal sources are also sometimes used.)
Upon action by the rennin, the milk is converted into a cheese curd and whey. The activity of the rennin is enhanced or catalyzed by both heat and lactic acid. Generally, lactic acid is supplied by lactic acid-producing bacteria, such as Streptococcus lactis and Streptococcus cremoris. Such bacteria feed primarily on the lactose in milk to produce the acid needed in the manufacture of cheese.
The development of acid is critical to the success of the entire cheesemaking process. Failure to develop acid properly can result in a soft, high moisture and high pH curd, and can further result in the development of gas, as well as fruity, fermented, or other undesirable flavors. Too much acid can result in a curd which is discolored or which has acidic or bitter flavors, body defects, or a relatively low moisture content. Hence, the proper production of acid through the development of a proper lactic bacteria culture is critical to the successful manufacture of cheese.
A bulk culture of lactic acid-producing bacteria is typically prepared in a vessel known as a bulk culture tank and serves as an inoculant for the milk to be made into cheese. This bulk culture of lactic bacteria generally comprises from between about 0.1% to 7% or more of the total volume of milk to be inoculated. Once a satisfactory lactic bacteria bulk culture has been prepared in the bulk culture tank, the bulk culture is introduced into a cheesmaking vessel containing the milk. The rennin enzyme is also added to the milk in the cheesemaking vessel, and the lactic bacteria cultures propagate while producing the necessary acid to aid the enzyme in producing cheese.
Most lactic acid-producing bacteria (such as Streptococcus lactis and Streptococcus cremoris) can exist in at least two different forms or phenotypes: proteinase positive (hereinafter sometimes referred to as "Prt+") and proteinase negative (hereinafter sometimes referred to as "Prt-"). The Prt+ lactic bacteria are characterized by an external, cell wall associated, proteinase enzyme system which breaks down casein (and perhaps other proteins) in the milk to simple peptides which are in part consumed during the propagation of the Prt+ lactic bacteria and which can create bitter flavors in the cheese. The Prt- lactic bacteria, on the other hand, are characterized by the absence of such an external, cell wall associated, proteinase enzyme system.
The propagation of Prt+ lactic bacteria has been found to be plasmid-linked, and upon loss of the proteinase-expressing plasmid, the Prt+ lactic bacteria produce Prt- daughter cells. Generally, variant proteinase negative lactic bacteria are produced from the proteinase positive lactic bacteria 1-2% of the time. It should be noted, however, that once the Prt+ lactic bacteria have lost the proteinase-expressing plasmid to produce Prt- lactic bacteria, the Prt- lactic bacteria are subsequently incapable of producing Prt+ lactic bacteria. Thus, introduction of a Prt- lactic bacteria phenotype into a cheesemaking system will result in the propagation of both Prt+ and Prt- lactic bacteria phenotypes; however, the introduction of a Prt- lactic bacteria phenotype into a cheesemaking system will result in the propagation of only the Prt- lactic bacteria phenotype.
In choosing a suitable bacterial strain for use in making cheese, the traditional rule has been that those bacterial strains which are unable to coagulate milk at room temperature in an eighteen to twenty-four hour period are unsuitable. Since, under normal conditions, Prt- lactic bacteria cannot coagulate milk within the traditional twenty-four hour period, the exclusive use of Prt- lactic bacteria for the making of cheese has been completely avoided in the prior art processes. Indeed, the prior art processes have taught away from the use of Prt- lactic bacteria in making cheese and have sought to suppress the production of Prt- lactic bacteria so as to maintain an active culture of Prt+ lactic bacteria.
Thus, while the prior art has recognized that some Prt- lactic bacteria may be employed, the prior art processes have generally sought to minimize the amount of Prt- lactic bacteria used. From the foregoing, it will be appreciated that the prior art processes are concerned mainly with methods for preparing lactic bacteria bulk cultures and for making cheese employing solely or primarily Prt+ lactic bacteria.
Although proteinase positive lactic bacteria have been preferred in the prior art cheesemaking processes, significant problems are inherent in the preparation and use of such Prt+ lactic bacteria cultures. For example, as mentioned above, the proteinase system of Prt+ lactic bacteria has been linked to the occurrence of bitter flavors in the resulting cheese. As the proteinase system breaks down casein in the milk, the resultant simple peptides cause a bitter flavor. Similarly, the proteinase system of the Prt+ lactic bacteria breaks down a considerable amount of casein in the milk which is consumed in the propagation of the Prt+ lactic bacteria, thereby significantly reducing the content of casein in the resultant cheese. The protein content of the resultant cheese is further reduced by the fact that the broken down or solubilized casein not consumed by the growing lactic bacteria is lost in the whey exuded from the curd. Thus, because of the reduction in protein content, the nutritional value of the cheese is correspondingly reduced.
Additionally, as mentioned above, introduction of a pure Prt+ lactic bacteria phenotype into a cheesemaking operation soon results in a mixture of both Prt+ and Prt- lactic bacteria phenotypes, thus introducing complex variables into the strategy of strain selection and propagation. Since the exact ratio of Prt+ to Prt- lactic bacteria will vary with time, it is difficult to control the production of Prt+ lactic bacteria. The result is that it is difficult to control the production of acid and thus to predict just how long it will take to produce the cheese.
Moreover, the ratio of Prt+ to Prt- lactic bacteria in the Prt+/Prt- lactic bacteria blend of the prior art processes may ultimately decrease to the point that the blend becomes incapable of producing lactic acid at a rate fast enough to carry out the cheesemaking process. This is due to the fact that in the prior art processes where Prt+/Prt- lactic bacteria blends occur, the growth of the Prt- lactic bacteria depends on the ability of the Prt+ lactic bacteria to break down casein and thereby produce sufficient nitorgenous nutrients for such growth. Thus, when the Prt+/Prt- lactic bacteria ratio decreases to the point that growth of the Prt- lactic bacteria is significantly inhibited, lactic acid production is also inhibited, and it becomes necessary to reisolate the Prt+ lactic bacteria to restore the acid production rate.
Other disadvantages of cheesemaking operations employing Prt+ lactic bacteria culture systems stem from the fact that significant growth of the Prt+ lactic bacteria in these systems is encouraged and carried out in the cheesemaking vessel and that the growth of such Prt+ lactic bacteria is relatively rapid. For example, careful control over the production of acid by the propagating Prt+ lactic bacteria in the cheesemaking vessel is made difficult because of the relatively rapid rate at which the Prt+ lactic bacteria in the cheesemaking vessel reproduce. Moreover, since the critical production of acid is largely dependent on the substantial initial growth of the Prt+ lactic bacteria in the cheesemaking vessel, conditions within the cheesemaking vessel must be carefully controlled so as to promote initial growth of the Prt+ lactic bacteria and carefully control the growth of the Prt+ lactic bacteria thereafter. Thus, the temperature in, for example, a Cheddar cheesemaking vessel must be raised gradually from 31.degree. C. to 38.degree. C. to optimize initial growth of the Prt+ lactic bacteria and to control the later growth thereof. Such a gradual temperature rise results in a relatively slow production of cheese; the required period of time for making Cheddar cheese using Prt+ lactic bacteria is typically from about three to about five hours from culture addition to salting of the curd.
The relatively long periods of time required to make cheese using Prt+ lactic bacteria and the substantial growth of the Prt+ lactic bacteria accomplished in the cheesemaking vessel result in perhaps the most significant problem associated with the use of Prt+ bacteria--the occurrence and growth of a significant number of bacteriophage in the milk within the cheesemaking vessel. These bacterial viruses are introduced into the cheesemaking operation when lactic bacteria infected with such viruses are present in the bulk culture inoculant or the surrounding cheesemaking environment. Since bacteriophage are reproduced in growing bacteria cells, the growth of bacteriophage is directly dependant upon the growth of the host bacteria. Bacteriophage growth is further enhanced by the presence of calcium ions in the surrounding environment. The prior art has attempted to protect against bacteriophage growth in the bulk culture tank rather than in the cheesemaking vessel, on the theory that lower concentrations of bacteriophage in the bulk culture inoculant would correspondingly reduce the number of bacteriophage in the milk within the cheesemaking vessel.
Generally speaking, bacteriophage populations of 10.sup.7 to 10.sup.8 or more bacteriophage plaque forming units per milliliter (pfu/ml) in the resulting whey at the end of the cheesemaking process indicate that there is enough bacteriophage in the cheesemaking operation to render the lactic bacteria culture substantially less effective, if not ineffective. Thus, such bacteriophage populations are considered unacceptable. Unfortunately, this critical limit is often approached or exceeded in the prior art processes using Prt+ lactic bacteria since there is relatively rapid growth of Prt+ lactic bacteria, and thus of bacteriophage, within the cheesemaking vessel.
Moreover, the large number of bacteriophage present in the resulting whey within the cheesemaking vessel has discouraged those skilled in the art from recycling the whey for use in the bulk culture tank in the preparation of other lactic bacteria bulk culture systems. Thus, the problems of bacteriophage in both the bulk culture tank and the cheesemaking vessel have plagued the cheese industry since its incipiency.
From the foregoing, it will be appreciated that what is needed in the art is a method for preparing a lactic bacteria bulk culture and a method for making fermented dairy products (such as cheese) wherein bacteriophage problems are effectively eliminated. Moreover, it would be a significant advancement in the art to provide a method for preparing a lactic bacteria bulk culture and a method for making fermented dairy products wherein bitter flavors are eliminated from the resulting fermented dairy product. Also, it would be a significant advancement in the art to provide a method for preparing a lactic bacteria bulk culture and a method for making fermented dairy products wherein a greater amount of the casein and other proteins in the milk are preserved and transferred to the resultant fermented dairy product. It would be a further advancement in the art to provide a method for preparing a lactic bacteria bulk culture and a method for making fermented dairy products wherein a single phenotype of lactic bacteria is involved, thereby eliminating complex variables in the selection and propagation of bacterial strains. Additionally, it would be a significant advancement in the art to provide a process for making fermented dairy products such as cheese wherein the production of acid is not dependent upon the growth of the lactic bacteria in the cheesemaking vessel, enabling the production of acid to be more carefully controlled. Finally, it would be a significant advancement in the art to provide a process for making fermented dairy products such as cheese wherein the initial temperature in the cheesemaking vessel may be raised higher than those initial temperatures presently experienced in the prior art processes, thereby reducing the required period of time for making the cheese and thereby inhibiting the growth of bacteriophage. Such novel methods are disclosed and claimed herein.